Combinations of sesquiterpene lactones and ditepene triepoxide lactones for synergistic inhibition of cyclooxygenase-2

ABSTRACT

A novel formulation is provided that serves to inhibit the inflammatory response in animals. The formulation comprises, as a first component an effective amount of a diterpene triepoxide lactone species and an effective amount of a second component of a sesquiterpene lactone species or derivatives thereof, and provides synergistic anti-inflammatory effects in response to physical or chemical injury or abnormal immune stimulation due to a biological agent or unknown etiology.

RELATED APPLICATIONS AND PRIORITY CLAIM

This application claims the benefit of U.S. Provisional ApplicationNo.60/222,167 filed Aug. 1, 2000.

FIELD OF THE INVENTION

The present invention relates generally to a composition exhibitingsynergistic inhibition of the expression and/or activity of induciblecyclooxygenase-2 (COX-2). More particularly, the composition comprises acombination of a diterpene triepoxide lactone species and asesquiterpene lactone species or derivatives thereof. The compositionfunctions synergistically to the activities of the individualcomponents, i.e., inhibiting the inducibility and/or activity ofinducible cyclooxygenase (COX-2) with no significant effect onconstitutive cyclooxygenase (COX-1).

BACKGROUND OF THE INVENTION

Inflammatory diseases affect more than fifty million Americans. As aresult of basic research in molecular and cellular immunology over thelast ten to fifteen years, approaches to diagnosing, treating andpreventing these immunologically-based diseases has been dramaticallyaltered. One example of this is the discovery of an inducible form ofthe cyclooxygenase enzyme. Constitutive cyclooxygenase (COX), firstpurified in 1976 and cloned in 1988, functions in the synthesis ofprostaglandins (PGs) from arachidonic acid (AA). Three years after itspurification, an inducible enzyme with COX activity was identified andgiven the name COX-2, while constitutive COX was termed COX-1.

COX-2 gene expression is under the control of pro-inflammatory cytokinesand growth factors. Thus, the inference is that COX-2 functions in bothinflammation and control of cell growth. While COX-2 is inducible inmany tissues, it is present constitutively in the brain and spinal cord,where it may function in nerve transmission for pain and fever. The twoisoforms of COX are nearly identical in structure but have importantdifferences in substrate and inhibitor selectivity and in theirintracellular locations. Protective PGs, which preserve the integrity ofthe stomach lining and maintain normal renal function in a compromisedkidney, are synthesized by COX-1. On the other hand, PGs synthesized byCOX-2 in immune cells are central to the inflammatory process.

The discovery of COX-2 has made possible the design of drugs that reduceinflammation without removing the protective PGs in the stomach andkidney made by COX-1. These selective COX-2 inhibitors may not only beanti-inflammatory, but may also be actively beneficial in the preventionand treatment of colon cancer and Alzheimer's disease.

An ideal formulation for the treatment of inflammation would inhibit theinduction and activity of COX-2 without affecting the activity of COX-1.Historically, the non-steroidal and steroidal anti-inflammatory drugsused for treatment of inflammation lack the specificity of inhibitingCOX-2 without affecting COX-1. Therefore, most anti-inflammatory drugsdamage the gastrointestinal system when used for extended periods. Thus,new treatments for inflammation and inflammation-based diseases areurgently needed.

The natural pharmacopoeia of plants and herbs used in traditionalmedicines for the treatment of inflammatory conditions was recentlyfound to contain COX-2 inhibitors. One such plant is Triptergiumwilfordi (TW). This herb, known as Lei Gong Teng in China, has been usedto treat patients suffering with rheumatoid arthritis with a 92%efficacy rate. Lei Gong Teng is available in the U.S. and is advertisedto support the healthy functioning of bone joints (www.China-Med.net).

Over 60 compounds have been isolated from TW, and many have beenidentified as having anti-inflammatory and immunosuppressive activity.Representative compounds that have been isolated from TW includetriptolide, 16-hydroxytriptolide, triptophenolide, tripdiolide, andcelastrol. However, the administration and therapeutic effectiveness ofthese compounds have generally been limited by their low margins ofsafety.

Triptolide is one of the active, nonalkaloid principles isolated from TWand possesses an extensive suppressive effect on immune function,especially on T and B lymphocytes. Structurally, triptolide is a memberof the group of diterpene triepoxide lactones (FIG. 1). The inhibitoryeffect is direct and believed to occur through the inhibition ofinterluken-2 (IL-2) production and IL-2R (receptor) expression (Tao, etal. (1995) J. Pharmacol. Exp. Therap. 272:1305; U.S. Pat. No. 5,500,340to Lipsky et al. Mar. 19, 1996). Clinical trials show that itsignificantly inhibits the proliferation of peripheral blood mononuclearcells of rheumatic arthritis patients. After receiving this medication,patients usually indicate that stiffness, walking, and hand strength areimproved with a decrease in inflammation index. Although not generallylife-threatening, adverse effects of triptolide are relatively common inthe clinical setting. Approximately 28% of patients taking this compoundshow some type of side effects, such as gastrointestinal disturbance,nausea and vomiting, hypotension and edema.

Therefore, while triptolide may be useful as an anti-inflammatory agent,it can be toxic even in clinically effective doses. Other researchershave used the triptolide molecule as a starting point for the synthesisof novel analogs expressing similar immune effects, while exhibitinglower toxicity (U.S. Pat. No. 5,962,516 to Qi et al. Oct. 5, 1999).Rather than modifying the triptolide molecule to achieve greaterefficacy and lower toxicity, it is the object of this invention tocombine triptolide, or a representative diterpene epoxide lactone, witha second molecule to produce a synergistic effect in the target cell.One such synergistic response would be the inhibition of inducibleCOX-2.

Leaves or infusions of feverfew, Tanacetum parthenium, have long beenused as a folk remedy for the relief of fever, arthritis and migraineheadaches. Previous reports using feverfew extracts have suggestedinterference with arachidonate metabolism as the mechanism behind thesepharmacological effects. In one study (Sumner et al. (1992) Biochem.Pharmacol. 43:2313-2320), crude chloroform extracts of fresh feverfewleaves produced dose-dependent inhibition of the generation ofthromboxane B2 and leukotriene B4 by ionophore- andchemoattractant-stimulated rat peritoneal leukocytes and humanpolymorphonuclear leukocytes. Other research has suggested inhibition ofplatelet aggregation and the platelet release reaction by feverfewextracts (Groenewegen et al. (1986) J. Pharm. Pharmacol. 38:709-712).Numerous publications suggest that the biologically active components offeverfew are sesquiterpene lactones, with parthenolide being the mostabundant.

In the literature approximately 25, separate biological effects havebeen reported for parthenolide. The potential pharmacological activitiesrange from the inhibition of isolated bovine prostaglandin synthetase(Pugh and Sambo (1988) J. Pharm. Pharmacol. 40:743-745) to theprevention of ethanol-induced gastric ulcers in the rat (Tournier et al.(1999) J. Pharm. Pharmacol. 51:215-219). Research at the molecular levelhas described parthenolide inhibition of nuclear factor kappa B (NF-kB)activation in several cell-based systems (Hehner et al. (1999) J.Immunol. 163:5617-5623; Bork et al. (1997) FEBS Letters 402:85-90) andinhibition of inducibile nitric oxide gene expression in cultured rataortic smooth muscle cells (Wong and Menendez (1999) Biochem. Biophys.Res. Commun. 262:375-380). While these molecular events may account, inpart, for some of the biological actions of parthenolide, there existsno consensus on the exact nature of the underlying mechanism for itsanti-inflammatory effects.

Clinically effective doses of parthenolide for migraine prevention areon the order of micrograms per kg body weight daily. Human clinicaltrials have verified the minimum effective dose for migraine prevention,as well as the associated discomfort of nausea and vomiting associatedwith use of 125 mg of feverfew extract per day. The feverfew extractsused in these trials generally contained between 0.2 to 0.7 percentparthenolide. Therefore, the minimally effective dose of parthenolidewould be estimated to be approximately 250 micrograms per day or 4micrograms parthenolide per kg body weight. Commercial, standardizedpreparations of feverfew deliver between 600 to 4000 microgramsparthenolide per daily dose. While more than sufficient to effectivelycontrol migraine frequency, it is doubtful that these doses ofparthenolide would be sufficient to address inflammatory responses.

Research literature on the in vitro anti-inflammatory effects ofparthenolide reports inhibitory constants in the micromolar range.Assuming a volume of distribution greater than several hundred mL per kgand a median resonance time less than 12 hours, these parthenolideconcentrations could only be achieved and maintained in vivo with dosingmg amounts of parthenolide per kg bodyweight. While such dosing studieshave been performed successfully in laboratory animals, no clinicalreports describe similar doses of parthenolide in humans. Based uponthese estimates, a clinically successful preparation of parthenolide forinflammatory conditions would be required to deliver at least 15 mgparthenolide/kg-day. However, such relatively high doses of parthenolidewould be commercially prohibitive due to the cost of production, evenfor a therapeutic formulation.

Combinations of botanicals containing triptolide along with other herbshave been use in both traditional and commercial medicine. However, thetriptolide content of TW is only 0.1%, leaving 99.9% of the ingredientsof TW as undefined. Such a large unknown fraction makes it extremelyunlikely that triptolide is a significant factor in the pharmacologicalresponse of TW in this formulation. Thus, it would be useful to identifya compound that would specifically enhance the anti-inflammatory effectof triptolide so that it could be used at sufficiently low doses or atcurrent clinical doses with no adverse side effects. The optimalformulation of triptolide for preserving the health of joint tissues,for treating arthritis or other inflammatory conditions has not yet beendiscovered. A formulation combining triptolide and parthenolide tosynergistically inhibit COX-2 and support the normalization of jointfunction has not yet been described or discovered.

While glucosamine is generally accepted as being effective and safe fortreating osteoarthritis, medical intervention into the treatment ofdegenerative joint diseases is generally restricted to the alleviationof its acute symptoms. Medical doctors generally utilize non-steroidaland steroidal anti-inflammatory drugs for treatment of osteoarthritis.These drugs, however, are not well-adapted for long-term therapy becausethey not only lack the ability to promote and protect cartilage, theycan actually lead to degeneration of cartilage or reduction of itssynthesis. Moreover, most non-steroidal, anti-inflammatory drugs damagethe gastrointestinal system when used for extended periods. Thus, newtreatments for arthritis are urgently needed.

The joint-protective properties of glucosamine would make it anattractive therapeutic agent for osteoarthritis except for twodrawbacks: (i) the rate of response to glucosamine treatment is slowerthan for treatment with anti-inflammatory drugs, and (ii) glucosaminemay fail to fulfill the expectation of degenerative remission. Instudies comparing glucosamine with non-steroidal anti inflammatoryagents, for example, a double-blinded study comparing 1500 mgglucosamine sulfate per day with 1200 mg ibuprofen, demonstrated thatpain scores decreased faster during the first two weeks in the ibuprofenpatients than in the glucosamine-treated patients. However, thereduction in pain scores continued throughout the trial period inpatients receiving glucosamine and the difference between the two groupsturned significantly in favor of glucosamine by week eight. Lopes Vaz,A., Double-blind clinical evaluation of the relative efficacy ofibuprofen and glucosamine sulphate in the management of osteoarthritisof the knee in outpatients, 8 Curr. Med Res Opin. 145-149 (1982). Thus,glucosamine may relieve the pain and inflammation of arthritis at aslower rate than the available anti-inflammatory drugs.

An ideal formulation for the normalization of cartilage metabolism ortreatment of osteoarthritis would provide adequate chondroprotectionwith potent anti-inflammatory activity. The optimal dietary supplementfor osteoarthritis should enhance the general joint rebuilding qualitiesoffered by glucosamine and attenuate the inflammatory response withoutintroducing any harmful side effects. It should be inexpensivelymanufactured and comply with all governmental regulations.

However, the currently available glucosamine formulations have not beenformulated to optimally attack and alleviate the underlying causes ofosteoarthritis and rheumatoid arthritis. Moreover, as with manycommercially-available herbal and dietary supplements, the availableformulations do not have a history of usage, nor controlled clinicaltesting, which might ensure their safety and efficacy.

It would be useful to provide a composition that would specifically andsynergistically enhance the anti-inflammatory effect of triptolide sothat these could be used at sufficiently low doses or at currentclinical doses with no adverse side effects.

SUMMARY OF THE INVENTION

The present invention provides a composition comprising a diterpenetriepoxide lactone species and a sesquiterpene lactone species thatspecifically and synergistically enhance the anti-inflammatory effect ofthe diterpene triepoxide lactone species or the sesquiterpene lactonespecies. Any diterpene triepoxide lactone or sesquiterpene lactonespecies is inclusive of derivatives of the respective genus. However,additional species or mixtures of species within the various genera maybe present in the composition which is limited in scope only by thecombinations of species within the various genera that exhibit theclaimed synergistic functionality.

The composition functions synergistically to inhibit the inducibilityand/or activity of COX-2 with no effect on COX-1.

The present invention further provides a composition of matter toincrease the rate at which glucosamine or chondrotin sulfate function tonormalize joint movement or reduce the symptoms of osteoarthritis.

One specific embodiment of the present invention is a compositioncomprising an effective amount of triptolide and parthenolide, whichfunctions synergistically to the activities of the individualcomponents.

The present invention further provides is a method of dietarysupplementation and a method of treating inflammation orinflammation-based diseases in a warm-blooded animal which comprisesproviding to the animal suffering symptoms of inflammation thecomposition of the present invention which specifically andsynergistically enhances the anti-inflammatory effect of a diterpenetriepoxide lactone, or a sesquiterpene lactone, and continuing toadminister such a dietary supplementation of the composition until saidsymptoms are eliminated or reduced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the general chemical structure of [A1] the diterpenetriepoxide lactone genus and [A2] triptolide as a species within thatgenus.

FIG. 2, [A1] and [A2] respectively, illustrates the general chemicalstructures of the sequuiterpene lactone genus and parthenolide as aspecies within that genus.

FIG. 3 provides a schematic for the experimental design of EXAMPLE 1.

FIG. 4 is a line graph depicting the percent inhibition of COX-2 enzymeprotein expression by individual and the combinations of the testedmaterials, as described in EXAMPLE 1, in the absence and presence ofarachidonic acid (AA).

DETAILED DESCRIPTION OF THE INVENTION

Before the present composition and methods of making and using thereofare disclosed and described, it is to be understood that this inventionis not limited to the particular configurations, as process steps, andmaterials may vary somewhat. It is also intended to be understood thatthe terminology employed herein is used for the purpose of describingparticular embodiments only and is not intended to be limiting since thescope of the present invention will be limited only by the appendedclaims and equivalents thereof.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

The present invention provides a composition having a synergisticinhibitory effect on the expression and/or activity of COX-2. Moreparticularly, the composition comprises an active diterpene triepoxidelactone and an active sesquiterpene lactone or derivatives thereof.Preferably, the molar ratio of the active diterpene triepoxide lactoneand active sesquiterpene lactone or derivatives thereof is within arange of 1:1 to 1:10, and more preferably within a range of 1:2.5 to1:10. The composition provided by the present invention can beformulated as a dietary supplement or therapeutic composition. Thecomposition functions synergistically to inhibit the inducibility and/oractivity of COX-2 with little or no effect on COX-1.

As used herein, the term “dietary supplement” refers to compositionsconsumed to affect structural or functional changes in physiology. Theterm “therapeutic composition” refers to any compounds administered totreat or prevent a disease.

As used herein, the term “active sesquiterpene lactone” or “activediterpene triepoxide lactone” refers to a species within thesesquiterpene lactone or diterpene triepoxide lactone genera that iscapable of inhibiting the inducibility and/or activity of COX-2 whilehaving no significant effect on COX-1 or is capable of inhibiting orreducing the severity of a severe inflammatory response. All activesequesterpene lactone species have an α-methylene or γ-lactonefunctional group and are capable of inhibiting or reducing the severityof an inflammatory response.

As used herein, diterpene triepoxide lactones, sesquiterpenes lactonesor “derivative” thereof refers to compounds or their naturally occurringor synthetic derivatives of species within the scope of the respectivegenera. Representative species within each genus are listed in Table 1.Of the species listed under each genus in Table 1, those containing atleast one asterisk (*) are preferred and those containing two asterisks(**) are particularly preferred.

TABLE 1 DITERPENE ACTIVE TRIEPOSIDES LACTONES SESQUITERPENE LACTONESTripchlorolide* 5-α-Hydroxy-dehydrocostuslacone Tripdiolide* Burrodin*Triptolide** Chlorochrymorin Triptonide** Chrysandiol Chrysartemin AChrysartermin B Cinerenin Confertiflorin* Costunolide* CurcoloneCynaropicrin Dehydrocostus lactone Dehydroleucodin Dehydrozaluzanin CDeoxylactucin Encelin** Enhydrin** Eremanthine EupaformoninEupaformosanin Eupatolide Furanodienone Helenalin* HeterogorgiolideLactucin Leucanthin B** Magnolialide Melapomdin A** MichelenolideParthenolide** Psilostachyin A* Repin

“Conjugates” of diterpene triepoxide lactones, sesquiterpenes lactonesor derivatives thereof means diterpene triepoxide lactones, orsesquiterpenes lactones covalently bound or conjugated to a memberselected from the group consisting of mono- or di-saccharides, aminoacids, sulfates, succinate, acetate and glutathione. Preferably, themono- or di-saccharides is a member selected from the group consistingof glucose, mannose, ribose, galactose, rhamnose, arabinose, maltose,and fructose.

Therefore, one preferred embodiment of the present invention is acomposition comprises a combination of an effective amount ofparthenolide and triptolide. The resulting formulation of thesecombination functions to synergistically inhibit the inducibility and/oractivity of COX-2 while showing little or no effect on COX-1. Therefore,the composition of the present invention essentially eliminates theinflammatory response rapidly without introducing any harmful sideeffects.

Preferably, the diterpene triepoxide lactones or triptolide (FIG. 1 [A1]and [A2]) employed in the present invention is a pharmaceutical gradebotanical extract such as can be obtained commercially, for example,from Folexco Flavor Ingredients, 150 Domorah Drive, Montogomeryville,Pa. 18936. The triptolide used can be readily obtained from Triptergiumwilfordiim. Pharmaceutical grade triptolide extract is standardized tohave a triptolide content of greater than 50 percent. Additionally, itcontains no alkaloids or glycosides normally found with triptolidegenerally isolated from botanical sources. The pharmaceutical, botanicalgrade extract must pass extensive safety and efficacy procedures. Asemployed in the practice of the present invention, the extract has aminimum triptolide content of about 1 to 50 percent by weight.Preferably, the minimum triptolide content is about 1 percent by weight.Alternatively, the triptolide may be synthesized using standardtechniques known in chemical synthesis.

The sesquiterpene lactone genus, as represented by FIG. 2, [A1], andspecifically the species parthenolide as represented by FIG. 2, [A2] ispreferably a pharmaceutical grade preparation such as can be obtainedfrom Folexco Flavor Ingredients, 150 Domorah Drive, Montogomeryville,Pa. 18936. The pharmaceutical grade extract must pass extensive safetyand efficacy procedures. Chrysanthemum parthenium or Tanacetum vulgareserve as ready sources of parthenolide. Pharmaceutical gradeparthenolide extract is greater than 5 weight percent. As employed inthe practice of the invention, the extract has a parthenolide content ofabout 5 to 95 percent by weight. Preferably, the minimum parthenolidecontent is greater than 50 percent by weight. Without limiting theinvention, it is anticipated that parthenolide would act to prevent anincrease in the rate of transcription of the COX-2 gene by thetranscriptional regulatory factor NF-kappa B.

The essence of the present invention is that, rather than modifying thediterpene triepoxide lactone or active sesquiterpene lactone moleculesto achieve greater efficacy and lower toxicity, a second component isadded that acts in a synergistic manner. Therefore, this inventionrelates to the discovery that when combining diterpene triepoxidelactones and an active sesquiterpene lactone or derivatives thereof, thecombination produces a synergistic effect of the activities of theindividual components in the target cell. One such synergistic responsewould be the specific inhibition of inducible COX-2.

It is discovered in the present invention that the combination ofditerpene triepoxide lactones and an active sesquiterpene lactoneprovides for a synergistic effect on the activity of diterpenetriepoxide lactones or an active sesquiterpene lactone. The result is amore selective effect on the activity of COX-2 at lower doses ofditerpene triepoxide lactones or an active sesquiterpene lactone. Bydecreasing the dose of diterpene triepoxide lactones or an activesesquiterpene lactone to achieve the desired COX-2 inhibition, theprobability of side effects from this compound decreases exponentially.

Preferably, a daily dose (mg/kg-day) of the present dietary supplementwould be formulated to deliver, per kg body weight of the animal, about0.001 to 3.0 mg diterpene triepoxide lactones and about 0.05 to 5 mgsesquiterpene lacotone. The composition of the present invention fortopical application would contain one of the following: about 0.001 to 1wt %, preferably 0.01 to 1 wt % diterpene triepoxide lactones orsesquiterpene lactones.

Additionally, the preferred composition of the present invention wouldproduce serum concentrations in the following range: 0.01 to 10 nMditerpene triepoxide lactones, and 0.001 to 10 μM sesquiterpenelacotone.

Table 2 below provides a list of diseases in which COX-2 enzymeexpression and activity may play a significant role and therefore areappropriate targets for normalization or treatment by the invention.

TABLE 2 Disease Tissue Affected Addison's Disease Adrenal AllergiesInflammatory cells Alzheimer Disease Nerve cells Arthritis Inflammatorycells Atherosclerosis Vessel wall Colon Cancer Intestine Crohn's DiseaseIntestine Diabetes (type I)/type II Pancreas Eczema Skin/Inflammatorycells Graves' Disease Thyroid Guillain-Barre Syndrome Nerve cellsInflammatory Bowel Disease Intestine Leukemia Immune cells LymphomasImmune cells Multiple Sclerosis Nerve cells Myasthenia GravisNeuromuscular junction Osteoarthritis Joint lining Psoriasis SkinPrimary Biliary Cirrhosis Liver Rheumatoid Arthritis Joint lining SolidTumors Various Systemic Lupus Erythematosis Multiple tissues Uveitis Eye

In addition to the combination of diterpene triepoxide lactones andsesquiterpene lactones, the present composition for dietary applicationmay include various additives such as other natural components ofintermediary metabolism, vitamins and minerals, as well as inertingredients such as talc and magnesium stearate that are standardexcipients in the manufacture of tablets and capsules.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, isotonic and absorptiondelaying agents, sweeteners and the like. These pharmaceuticallyacceptable carriers may be prepared from a wide range of materialsincluding, but not limited to, diluents, binders and adhesives,lubricants, disintegrates, coloring agents, bulking agents, flavoringagents, sweetening agents and miscellaneous materials such as buffersand absorbents that may be needed in order to prepare a particulartherapeutic composition. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredients, its use in the present composition is contemplated.In one embodiment, talc and magnesium stearate are included in thepresent formulation. When these components are added they arepreferably, the Astac Brand 400 USP talc powder and the veritable gradeof magnesium stearate. Other ingredients known to affect the manufactureof this composition as a dietary bar or functional food can includeflavorings, sugars, amino-sugars, proteins and/or modified starches, aswell as fats and oils.

The dietary supplements, lotions or therapeutic compositions of thepresent invention can be formulated in any manner known by one of skillin the art. In one embodiment, the composition is formulated into acapsule or tablet using techniques available to one of skill in the art.In capsule or tablet form, the recommended daily dose for an adult humanor animal would preferably be contained in one to six capsules ortablets. However, the present compositions may also be formulated inother convenient forms, such as an injectable solution or suspension, aspray solution or suspension, a lotion, gum, lozenge, food or snackitem. Food, snack, gum or lozenge items can include any ingestableingredient, including sweeteners, flavorings, oils, starches, proteins,fruits or fruit extracts, vegetables or vegetable extracts, grains,animal fats or proteins. Thus, the present compositions can beformulated into cereals, snack items such as chips, bars, gum drops,chewable candies or slowly dissolving lozenges.

The present invention contemplates treatment of all types ofinflammation-based diseases, both acute and chronic. The presentformulation reduces the inflammatory response and thereby promoteshealing of, or prevents further damage to, the affected tissue. Apharmaceutically acceptable carrier may also be used in the presentcompositions and formulations.

According to the present invention, the animal may be a member selectedfrom the group consisting of humans, non-human primates, such as dogs,cats, birds, horses, ruminants or other warm blooded animals. Theinvention is directed primarily to the treatment of human beings.Administration can be by any method available to the skilled artisan,for example, by oral, topical, transdermal, transmucosal, or parenteralroutes.

The following examples are intended to illustrate but not in any waylimit the invention:

EXAMPLE 1 Inhibition of Cyclooxygenase-2 Enzyme Expression in Human TCells by Triptolide and Parthenolide

This example hypothetically illustrates the effect of triptolide andparthenolide on COX-2 in cultured Jurkat cells. It is found thattriptolide alone may decrease the expression of COX-2 protein in PMAstimulated cells and that parthenolide has little effect in thedose-range tested. In the presence of arachidonic acid (AA), theeffectiveness of triptolide is markedly reduced. However, a combinationof the two compounds exerted a powerful inhibition of the expression ofCOX-2 in the presence and absence of AA, with no observable signs oftoxicity.

Chemicals: Anti-COX-2 antibodies may be purchased from UpstateBiotechnology (Lake Placid, N.Y.). Triptolide and parthenolide may beobtained from Sigma (St. Louis, Mo.). Arachidonic acid (AA), PMA and allother chemical may also be purchased from Sigma and are of the highestpurity commercially available.

Human T cell lines: The Jurkat cell line is useful as a model for humanT cells and may be obtained from the American Type Culture Collection(Bethesda, Md.). COX-2 is inducible in the Jurkat cell by PMA.

Cell plating: The Jurkat cells are propagated in suspension according tothe instructions of the supplier. For experimentation, cells are seededfrom a log-phase culture at a density of 1×10⁵ cells per mL in 100 mmplates, 20 mL per plate, 3 plates per treatment. Serum concentration inthe test medium is maintained at 0.5%. After 24 hours, thephytohemagglutinin (PHA) or PHA/AA combinations are added to the cellcultures, in 10 μL aliquots, to achieve effective concentrations.

Gel Electrophoresis: Sodium dodecyl sulfate polyacryamide gelelectrophoresis (PAGE) is performed using 10% polyacrylamide gels asdescribed by Laemmli, U. K. and Favre, M. (J. Mol. Biol. (1973) 80:575)with the modification that the cell lysates (100 μg/lane) are heated at100 ° C. for three minutes.

Immunoblotting: The immunoblotting is performed as described by Tobin etal. (Proc. Nat. Acad. Sci. USA (1979) 76:4350), however, Milliblot SDEelectroblot apparatus (Millipore, Bedford, Mass.) is used to transferproteins from the polyacrylamide gels to an Immobilon® membrane filter.Complete transfers are accomplished in 25-30 minutes at 500 mA.Membranes used for blotting are blocked by incubating in TBS (Trisbuffered saline, 50 mM Tris, 150 mM NaCl, pH 7.5) containing 5% nonfatdry milk for 30 minutes at room temperature. COX-2 protein is visualizedby incubation of the blots with the anti-COX-2 antibody in TBST (0.5%Tween 20 in TBS) for two hours and then a second incubation at roomtemperature with alkaline phosphatase-conjugated secondary antibodydiluted 1:1000 in TBST for two hours. The enzymatic reaction isdeveloped for 15 minutes. The molecular weight of COX-2 is estimated byadding a molecular weight standard to reference lanes and staining themembrane filters with amido black 10B.

Blots are translated into TIFF-formatted files with a Microtech 600GSscanner and quantified using Scan Analysis (BIOSOFT, Cambridge, UK).Summary scans are then printed and peak heights are measured directlyfrom the figure. One density unit (Du) is defined as one mm of theresulting peak height.

Protein determination: Spectrophotometric determination of proteinconcentration is determined with bicinchoninic acid as reported by Smithet al. (Anal. Biochem. (1985) 150:76).

FIG. 3 provides a schematic for the experimental design in which Jurkatcells are stimulated with PHA in the absence and present of arachidonicacid. Triptolide or parthenolide alone, or a combination of triptolideand parthenolide are added in a volume of 10 μL immediately to themedium immediately following the PHA treatment. Appropriate controlsreceive solvent only. Final concentrations of triptolide orparthenolideare 0, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0 and 10 nM. For the mixtures, thefirst seven doses are simply combined. For example, the first dose ofthe combined treatment contains 0.01 nM triptolide and 0.01 nM oleanolicacid. Twenty-four hours after treatment, the cells are harvested, lysedand western blotting is done for the determination of COX-2 proteinexpression.

FIG. 4 is a line graph depicting the percent inhibition of COX-2 enzymeprotein expression by triptolide, parthenolide and the combination oftriptolide with parthenolide in the absence and presence of arachidonicacid. It is observed that triptolide functions to inhibit the expressionof inducible cyclooxygenase 2 enzyme in the Jurkat cell line in theabsence of arachidonic acid, and that this activity is enhanced morethan 10-fold by parthenolide. Parthenolide alone does not inhibit COX-2expression at physiologically relevant doses. In the presence ofparthenolide, the inhibition of inducible COX-2 by triptolide is nearlycomplete, even at very low concentrations. In the presence ofarachidonic acid, triptolide inhibition of COX-2 enzyme protein iscompromised, but restored in the presence of parthenolide.

EXAMPLE 2 Normalization of Joint Functioning Following Trauma

A representative composition of the present invention as a dietarysupplement would be in an oral formulation, i.e. tablets, that wouldsupply 0.01 mg triptolide/kg per day and 5.0 mg parthenolide/kg per day.Normalization of joint movement following physical trauma due toexercise or repetitive movement stress would be expected to occurfollowing two to ten doses. This result would be expected in allanimals.

EXAMPLE 3 Clinical Effectiveness of Lotion Formulations in the Treatmentof Acne Rosacea

A lotion designed to contain 0.1% wt triptolide and 0.1% parthenolide isapplied to affected areas of patients who have exhibited acne rosace asdiagnosed by their own practitioner and confirmed by an independentboard-certified dermatologist. Self-evaluation tests and areadministered one week prior to the study to quantify the surface areaaffected and redness. In addition, similar variables are scored by theprofessional clinical staff not aware of the patients treatment status.These evaluations are repeated on Days 0, 7, 14 and 21.

Patients are randomly assigned to the test formulation or a placebo atthe start of the study. The test formulation and placebo are applied tothe affected area one or two times per day. Treatment for healthconditions such as diabetes, hypertension, etc. is allowed during thestudy. Scores are statistically compared between the test formulationand the placebo for each of the four observational periods. Patientstreated with the combination composition of the present invention in alotion formulation are considered improved if the patients' scoresimprove by greater than 20% from the pre-test scores within eachcategory evaluated. The percentage of persons exhibiting improvement arecompared between the combination formulations and the placebo control.The difference between the two groups is considered statisticallysignificant if the probability of rejecting the null hypothesis whentrue is less than five percent.

EXAMPLE 4 Clinical Effectiveness of Lotion Formulations in the Treatmentof Psoriasis

This example is performed in the same manner as described in the Example3, except that the composition is applied to affected areas of patientswho have exhibited psoriasis as diagnosed by their own practitioner andconfirmed by an independent board-certified dermatologist.Self-evaluation tests are administered one week prior to the study toquantify the surface area affected and skin condition. In addition,similar variables are scored by the professional clinical staff notaware of the patients treatment status. These evaluations are repeatedon Days 0, 7, 30 and 60.

Patients are randomly assigned to the test formulation or placebo at thestart of the study. The test formulation and placebo are applied to theaffected area one or two times per day. Treatment for health conditionssuch as diabetes, hypertension, etc. is allowed during the study. Scoresare statistically compared between the test formulation and the placebofor each of the four observational periods. Patients treated with thetriptolide/parthenolide lotion formulation are considered improved ifthe patients' scores improve by greater than 20% from the pre-testscores within each category evaluated. The percentage of personsexhibiting improvement is compared between the triptolide/parthenolideformulation and the placebo control. The difference between the twogroups is considered statistically significant if the probability ofrejecting the null hypothesis when true is less than five percent.

EXAMPLE 5 Clinical Effectiveness of an Oral Formulation in the Treatmentof Alzheimer's Disease

An oral formulation as described in the Example 2 is administered topatients who have manifested an early stage of Alzheimer's Disease (AD),as diagnosed by their own practitioner and confirmed by an independentboard-certified neurologist. Two weeks before the clinical trial, thepatients undergo appropriate psychoneurological tests such as the MiniMental Status Exam (MMSE), the Alzheimer Disease Assessment Scale(ADAS), the Boston Naming Test (BNT), and the Token Test (TT).Neuropsychological tests are repeated on Day 0, 6 weeks and 3 months ofthe clinical trial. The tests are performed by neuropsychologists whoare not aware of the patient's treatment regimen.

Patients are randomly assigned to the test formulation or placebo at thestart of the study. The test formulation and placebo are taken orallyone or two times per day. Treatment for conditions such as diabetes,hypertension, etc. is allowed during the study. Scores are statisticallycompared between the test formulation and the placebo for each of thethree observational periods. Without treatment, the natural course of ADis significant deterioration in the test scores during the course of theclinical trial. Patients treated with the triptolide/parthenolideformulation are considered improved if the patients' scores remain thesame or improve during the course of the clinical trial.

EXAMPLE 6 Clinical Effectiveness of an Oral Formulation in the Treatmentand Prevention of Colon Cancer

An oral formulation as described in Example 2 is administered topatients who have manifested an early stage of colon cancer as diagnosedby their own practitioner and confirmed by a independent board-certifiedoncologist.

Patients are randomly assigned to the test formulation or placebo at thestart of the study. The test formulation and placebo are taken orallyone or two times per day. Treatment for conditions such as diabetes,hypertension, etc. is allowed during the study. Endoscopic evaluationsare made at one, two, six and twelve months. Evidence of reappearance ofthe tumor during any one of the four follow-up clinical visits isconsidered a treatment failure. The percentage of treatment failures iscompared between the triptolide/parthenolide formulation and the placebocontrol. The difference between the two groups is consideredstatistically significant if the probability of rejecting the nullhypothesis when true is less than five percent.

EXAMPLE 7 Clinical Effectiveness of an Oral Formulation in the Treatmentof Irritable Bowel Syndrome

An oral formulation as described in Example 2 is administered topatients who have manifested irritable bowel syndrome as diagnosed bytheir practitioner. Normal bowel functioning is restored within 24hours.

EXAMPLE 8 Normalization of Joint Functioning in Osteoarthritis

Using a composition described in Example 2, normalization of jointstiffness due to osteoarthritis occurs following five to twenty doses,in the presence or absence of glucosamine or chondroitin sulfate. Inaddition, the composition does not interfere with the normal jointrebuilding effects of these two proteoglycan constituents, unliketraditional non-steroidal anti-inflammatory agents.

EXAMPLE 9 Inhibition of COX-2 Enzyme Production of Prostaglandin E2 inMurine B Cells by Parthenolide and Triptolide

This example illustrates the superior COX-2 inhibitory potency andselectivity of the combination of parthenolide and triptolide of thepresent invention compared to parthenolide or triptolide alone.

Inhibition of COX-2 Mediated Production of PGE2 in RAW 264.7 Cells

Equipment—balancer, analytical, Ohaus Explorer (Ohaus Model #EO1140,Switzerland), biosafety cabinet (Forma Model #F1214, Marietta, Ohio),pipettor, 100 to 1000 μL (VWR Catalog #4000-208, Rochester, N.Y.), cellhand tally counter (VWR Catalog #23609-102, Rochester, N.Y.), CO₂incubator (Forma Model #F3210, Marietta, Ohio), hemacytometer (HausserModel #1492, Horsham, Pa.), microscope, inverted (Leica Model #DM IL,Wetzlar, Germany), multichannel pipettor, 12-Channel (VWR Catalog#53501-662, Rochester, N.Y.), Pipet Aid (VWR Catalog #53498-103,Rochester, N.Y.), Pipettor, 0.5 to 10 μL (VWR Catalog #4000-200,Rochester, N.Y.), pipettor, 100 to 1000 μL (VWR Catalog #4000-208,Rochester, N.Y.), pipettor, 2 to 20 μL (VWR Catalog #4000-202,Rochester, N.Y.), pipettor, 20 to 200 μL (VWR Catalog #4000-204,Rochester, N.Y.), PURELAB Plus Water Polishing System (U.S. Filter,Lowell, Mass.), refrigerator, 4° C. (Forma Model #F3775, Marietta,Ohio), vortex mixer (VWR Catalog #33994-306, Rochester, N.Y.), waterbath (Shel Lab Model #1203, Cornelius, Oreg.).

Cells, Chemicals, Reagents and Buffers—Cell scrapers (Corning Catalog#3008, Corning, N.Y.), dimethylsulfoxide (DMSO) (VWR Catalog #5507,Rochester, N.Y.), Dulbecco's Modification of Eagle's Medium (DMEM)(Mediatech Catalog #10-013-CV, Herndon, Va.), fetal bovine serum, heatinactivated (FBS-HI) (Mediatech Catalog #35-011-CV, Herndon, Va.),lipopolysaccharide (LPS)(Sigma Catalog #L-2654, St. Louis, Mo.),microfuge tubes, 1.7 mL (VWR Catalog #20172-698, Rochester, N.Y.),penicillin/streptomycin (Mediatech Catalog #30-001-CI, Herndon, Va.),pipettips for 0.5 to 10 μL pipettor (VWR Catolog #53509-138, Rochester,N.Y.), pipet tips for 100-1000 μL pipettor (VWR Catolog #53512-294,Rochester, N.Y.), pipet tips for 2-20 μL and 20-200 μL pipettors (VWRCatolog #53512-260, Rochester, N.Y.), pipets, 10 mL (Becton DickinsonCatalog #7551, Marietta, Ohio), pipets, 2 mL (Becton Dickinson Catalog#7507, Marietta, Ohio, pipets, 5 mL (Becton Dickinson Catalog #7543,Marietta, Ohio), RAW 264.7 Cells (American Type Culture CollectionCatalog #TIB-71, Manassas, Va.), test compounds (liquid CO₂ hops extractfrom Hopunion, Yakima, Wash.), tissue culture plates, 96-well (BectonDickinson Catalog #3075, Franklin Lanes, N.J.), Ultra-pure water(Resistance=18 megaOhm-cm deionized water).

General Procedure—RAW 264.7 cells, obtained from ATCC, were grown inDMEM medium and maintained in log phase growth. The DMEM growth mediumwas made as follows: 50 mL of heat inactivated FBS and 5 mL ofpenicillin/streptomycin were added to a 500 mL bottle of DMEM and storedat 4° C. This was warmed to 37° C. in a water bath before use and forbest results should be used within three months.

On day one of the experiment, the log phase 264.7 cells were plated at8×10⁴ cells per well in 0.2 mL growth medium per well in a 96-welltissue culture plate. After 6 to 8 hours post plating, 100 μL of growthmedium from each well was removed and replaced with 100 μL fresh medium.A 1.0 mg/mL solution of LPS, which was used to induce the expression ofCOX-2 in the RAW 264.7 cells, was prepared by dissolving 1.0 mg of LPSin 1 mL DMSO. It was mixed until dissolved and stored at 4° C.Immediately before use, it was thawed at room temperature or in a 37° C.water bath.

On day two of the experiment, the test materials were prepared as 1000×stock in DMSO. For example, if the final concentration of the testmaterial was to be 10 μg/mL, a 10 mg/mL stock was prepared by dissolving10 mg of the test material in 1 mL of DMSO. Fresh test materials wereprepared on day 2 of the experiment. In 1.7 mL microfuge tubes, 1 mLDMEM without FBS was added to obtain test concentrations of 0.05, 0.10,0.5, and 1.0 μg/mL. 2 μL of the 1000× DMSO stock of the test materialwas added to the 1 mL of medium without FBS. The tube contained thefinal concentration of the test material was concentrated 2-fold. Thetube was placed in incubator for 10 minutes to equilibrate.

One-hundred mL of medium was removed from each well of the cell platesprepared on day one. One-hundred mL of equilibrated 2×finalconcentration the test compounds were added to cells and incubated for90 minutes. LPS in DMEM without FBS was prepared by adding 44 μL of the1 mg/ml DMSO stock to 10 mL of medium. For each well of cells to bestimulated, 20 μL of LPS (final concentration of LPS is 0.4 μg/mL ofLPS) was added. The LPS stimulation was continued for 24 hours, afterwhich the supernatant medium from each well was transferred to a cleanmicrofuge tube for determination of the PGE2 content in the medium.

Determination of COX-1 Enzyme Inhibition by Parthenolide andAndrographolide

The ability of a test material to inhibit COX-1 synthesis of PGE2 wasdetermined essentially as described by Noreen, Y., et al. (J. Nat. Prod.61, 2-7, 1998).

Equipment—balancer (2400 g, Acculab VI-2400, VWR Catalog #11237-300,Rochester, N.Y.), balancer, analytical, Ohaus Explorer (Ohaus Model#EO1140, Switzerland), biosafety cabinet (Forma Model #F1214, Marietta,Ohio), Freezer, −30° C. (Forma Model #F3797), Freezer, −80° C. Ultralow(Forma Model #F8516, Marietta, Ohio), heated stirring plate (VWR Catalog#33918-262, Rochester, N.Y.), ice maker (Scotsman Model #AFE400A-1A,Fairfax, S.C.), multichannel pipettor, 12-Channel (VWR Catalog#53501-662, Rochester, N.Y.), Multichannel Pipettor, 8-Channel (VWRCatalog #53501-660, Rochester, N.Y.), orbital shaker platform(Scienceware #F37041-0000, Pequannock, N.J.), pH meter (VWR Catalog#33221-010, Rochester, N.Y.), pipet aid (VWR Catalog #53498-103,Rochester, N.Y.), pipettor, 0.5 to 10 μL (VWR Catalog #4000-200,Rochester, N.Y.), pipettor, 100 to 1000 μL (VWR Catalog #4000-208,Rochester, N.Y.), pipettor, 2 to 20 μL (VWR Catalog #4000-202,Rochester, N.Y.), pipettor, 20 to 200 μL (VWR Catalog #4000-204,Rochester, N.Y.), PURELAB Plus Water Polishing System (U.S. Filter,Lowell, Mass.), refrigerator, 4° C. (Forma Model #F3775, Marietta,Ohio), vacuum chamber (Sigma Catalog #Z35, 407-4, St. Louis, Mo.),vortex mixer (VWR Catalog #33994-306, Rochester, N.Y.)

Supplies and Reagents—96-Well, round-bottom plate (Nalge Nunc #267245,Rochester, N.Y.), arachidonic acid (Sigma Catalog #A-3925, St. Louis,Mo.), centrifuge tubes, 15 mL, conical, sterile (VWR Catalog #20171-008,Rochester, N.Y.), COX-1 enzyme (ovine) 40,000 units/mg (Cayman ChemicalCatalog #60100, Ann Arbor, Mich.), dimethylsulfoxide (DMSO) (VWR Catalog#5507, Rochester, N.Y.), ethanol 100% (VWR Catalog #MK701908, Rochester,N.Y.), epinephrine (Sigma Catalog #E-4250, St. Louis, Mo.), glutathione(reduced) (Sigma Catalog #G-6529, St. Louis, Mo.), graduated cylinder,1000 mL (VWR Catalog #24711-364, Rochester, N.Y.), hematin (porcine)(Sigma catalog #H-3281, St. Louis, Mo.), hydrochloric acid (HCl) (VWRCatalog #VW3110-3, Rochester, N.Y.), Kim Wipes (Kimberly Clark Catalog#34256, Roswell, Ga.), microfuge tubes, 1.7 mL (VWR Catalog #20172-698,Rochester, N.Y.), NaOH (Sigma Catalog #S-5881, St. Louis, Mo.), pipettips for 0.5 to 10 μL pipettor (VWR Catolog #53509-138, Rochester,N.Y.),pipet tips for 100-1000 μL pipettor (VWR Catolog #53512-294,Rochester, N.Y.), pipet tips for 2-20 μL and 20-200 μL pipettors (VWRCatolog #53512-260, Rochester, N.Y.), prostaglandin E2 (Sigma Catalog#P-5640, St. Louis, Mo.), prostaglandin F2alpha (Sigma Catalog #P-0424,St. Louis, Mo.), stir bar, magnetic (VWR Catalog #58948-193, Rochester,N.Y.), storage bottle, 1000 mL (Corning Catalog #1395-1L, Corning,N.Y.), storage bottle, 100 mL (Corning Catalog #1395-100, Corning,N.Y.), CO₂ extract of hops (Hopunion, Yakima, Wash.), Tris-HCl (SigmaCatalog #T-5941, St. Louis, Mo.), ultra-pure water (Resistance=18megaOhm-cm deionized water).

General Procedure—Oxygen-free 1.0M Tris-HCl buffer (pH 8.0) was preparedas follows. In a 1000 mL beaker, 12.11 g Trizma HCl was dissolved into900 mL ultra-pure water. The beaker was placed on a stir plate with astir bar. NaOH was added until the pH reached 8.0. The volume wasadjusted to a final volume of 1000 mL and stored in a 1000 mL storagebottle.

The Tris-HCl buffer was placed into a vacuum chamber with the toploosened and the air pump was turned on until the buffer stoppedbubbling. The vacuum chamber was then turned off and the storage bottlewas tightly covered. This step was repeated each time when oxygen-freeTris-HCl buffer was used.

One mL cofactor solution was prepared by adding 1.3 mg (−) epinephrine,0.3 mg reduced glutathione and 1.3 mg hematin to 1 mL oxygen freeTris-HCl buffer. The solutions of the test material were prepared asneeded. i.e. 10 mg of aspirin was weighed and dissolved into 1 mL DMSO.

Enzymes, i.e. prostaglandin E2 or prostaglandin F2alpha, were dissolvedin oxygen free Tris-HCl buffer as follows, i.e. on ice, 6.5 μL of enzymeat 40,000 units/mL was taken and added to 643.5 μL of oxygen freeTris-HCl buffer. This enzyme solution is enough for 60 reactions. TheCOX-1 enzyme solution was prepared as follows: In a 15 mL centrifugetube, 10 μL COX-1 enzyme at 40,000 units/mL was added to oxygen freeTris-HCl with 50 μL of the cofactor solution per reaction. The mixturewas incubated on ice for 5 minutes. For 60 reactions, 650 μl enzyme wereadded in oxygen free Tris-HCl buffer with 3.25 mL cofactor solution.

Sixty microliters of the enzyme solution were combined with 20 μl of thetest solution in each well of a 96 well plate. Final concentrations ofthe test solutions were 100, 50, 25, 12.5, 6.25 and 3.12 μg/mL. Theplates were preincubated on ice for 10 minutes. Twenty μL arachidonicacid (30 μM) was added and incubated for 15 minutes at 37° C.

Two M HCl was prepared by diluting 12.1 N HCl. in a 100 mL storagebottle. 83.5 mL ultra-pure water was added and then 16.5 mL 12.1 N HClwas added. It was stored in a 100 mL storage bottle and placed in theBiosafty cabinet. The reaction was terminated by adding 10 μL 2 M HCl.The final solution was used as the supernatant for the PGE₂ assay.

Determination of PGE2 Concentration in Medium

The procedure followed was that essentially described by Hamberg, M. andSamuelson, B. (J. Biol. Chem. 1971. 246, 6713-6721); however acommercial, nonradioactive procedure was employed.

Equipment—freezer, −30° C. (Forma Model #F3797), heated stirring plate(VWR Catalog #33918-262, Rochester, N.Y.), multichannel pipettor,12-Channel (VWR Catalog #53501-662, Rochester, N.Y.), orbital shakerplatform (Scienceware #F37041-0000, Pequannock, N.J.), Pipet Aid (VWRCatalog #53498-103, Rochester, N.Y.), pipettor, 0.5 to 10 μL (VWRCatalog #4000-200, Rochester, N.Y.), pipettor, 100 to 1000 μL (VWRCatalog #4000-208, Rochester, N.Y.), pipettor, 2 to 20 μL (VWR Catalog#4000-202, Rochester, N.Y.), pipettor, 20 to 200 μL (VWR Catalog#4000-204, Rochester, N.Y.), plate reader (Bio-tek Instruments Model#Elx800, Winooski, Vt.), PURELAB Plus Water Polishing System (U.S.Filter, Lowell, Mass.), refrigerator, 4° C. (Forma Model #F3775,Marietta, Ohio).

Chemicals, Reagents and Buffers—Prostaglandin E₂ EIA Kit-Monoclonal480-well (Cayman Chemical Catalog #514010, Ann Arbor, Mich.), centrifugetube, 50 mL, conical, sterile (VWR Catalog #20171-178, Rochester, N.Y.),Dulbecco's Modification of Eagle's Medium (DMEM) (Mediatech Catalog#10-013-CV, Herndon, Va.), graduated cylinder, 100 mL (VWR Catalog#24711-310, Rochester, N.Y.), Kim Wipes (Kimberly Clark Catalog #34256,Roswell, Ga.), microfuge tubes, 1.7 mL (VWR Catalog #20172-698,Rochester, N.Y.), penicillin/streptomycin (Mediatech Catalog #30-001-CI,Herndon, Va.), pipet tips for 0.5 to 10 μL pipettor (VWR Catolog#53509-138, Rochester, N.Y.), pipet tips for 100-1000 μL pipettor (VWRCatolog #53512-294, Rochester, N.Y.), pipet tips for 2-20 μL and 20-200μL pipettors (VWR Catolog #53512-260, Rochester, N.Y.), pipets, 25 mL(Becton Dickinson Catalog #7551, Marietta, Ohio), storage bottle, 100 mL(Corning Catalog #1395-100, Corning, N.Y.), storage bottle, 1000 mL(Corning Catalog #1395-1L, Corning, N.Y.), ultra-pure water(Resistance=18 megaOhm-cm deionized water).

General Procedure—EIA Buffer was prepared by diluting the contents ofthe EIA Buffer Concentrate (vial #4) with 90 ml of Ultra-pure water.Vial #4 was rinsed several times to ensure all crystals had been removedand was then placed into a 100 mL storage bottle and stored at 4° C.

The Wash Buffer was prepared by diluting Wash Buffer Concentrate (vial#5) 1:400 with Ultra-pure water. 0.5 ml/liter of Tween 20 (vial #5a) wasthen added (using a syringe for accurate measurement). To prepare oneliter of Wash Buffer add 2.5 ml Wash Buffer Concentrate, 0.5 mlTween-20, and 997 ml Ultra-pure water. The solution was stored in a 1liter storage bottle at 4° C.

The Prostaglandin E₂ standard was reconstituted as follows. A 200 μLpipet tip was equilibrated by repeatedly filling and expelling the tipseveral times in ethanol. The tip was used to transfer 100 μL of thePGE₂ Standard (vial #3) into a 1.7 mL microfuge tube. 900 μl Ultra-purewater was added to the tube and stored at 4° C., which was stable for ˜6weeks. The Prostaglandin E₂ acetylcholinesterase tracer wasreconstituted as follows. 100 μL PGE₂ tracer (vial #2) was mixed with 30mL of the EIA Buffer in a 50 mL centrifuge tube and stored at 4° C.

The Prostaglandin E₂ monoclonal antibody was reconstituted as follows.100 μL PGE₂ Antibody (vial #1) was mixed with 30 mL of the EIA buffer ina 50 mL centrifuge tube and stored at 4° C.

DMEM with penicillin/streptomycin was prepared by adding 5 mLpenicillin/streptomycin into 500 mL DMEM and stored at 4° C.

The plates were set up as follows: Each plate contained a minimum of twoblanks (B), two non-specific binding wells (NSB), two maximum bindingwells (B₀), and an eight point standard curve run in duplicate (S1-S8).Each sample was assayed at a minimum of two dilutions and each dilutionwas run in duplicate.

The standard was prepared as follows: Eight 1.7 mL microuge tubes werelabeled as tubes 1-8. 900 μL DMEM into was added to tube 1 and 500 μLDMEM to tubes 2-8. 100 μL of the PGE₂ standard was added to tube 1 andmixed. Five-hundred mL of solution was taken from tube 1 and put intotube 2, and this process was repeated through tube 8.

Fifty mL EIA Buffer and 50 μl DMEM were added into the NSB wells. Fiftyμl DMEM was added to the B₀ wells. Fifty mL of solution was taken fromtube #8 and added to both the lowest standard wells (S8). Fifty mL wastaken from tube #7 and added to each of the next two wells. This wascontinued through to tube #1. (the same pipet tip was used for all 8 ofthe standards making sure to equilibrate the tip in each new standard bypipeting up and down in that standard. Using a P200, 50 μl of eachsample at each dilution was added to the sample wells.

Using a 12 channel pipetor, 50 μl of the Prostaglandin E₂acetylcholinesterase tracer was added to each well except the TotalActivity (TA) and the Blank (B) wells. Using the 12 channel pipetor, 50μl of the Prostaglandin E₂ monoclonal antibody was added to each wellexcept the Total Activity (TA), the (NSB), and the Blank (B) wells. Theplate was covered with plastic film (item #7) and incubated for 18 hoursat 4° C.

The plates were developed as follows: one 100 μL vial of Ellman'sReagent (vial #8) was reconstituted with 50 ml of Ultra-pure water in a50 mL centrifuge tube. It was protected from light and used the sameday. The wells were washed and rinsed five times with Wash Buffer usinga 12 channel pipettor. Two-hundred mL of Ellman's Reagent was added toeach well using a 12 channel pipettor and 5 μl of Tracer to the totalactivity (TA) wells was then added to each well using a P10 pipette. Theplate was covered with a plastic film and placed on orbital shaker inthe dark for 60-90 minutes.

The plate was read in the Bio-tek plate reader at a single wavelengthbetween 405 and 420 nm. Before reading each plate, the bottom was wipedwith a Kim wipe. The plate should be read when the absorbance of thewells is in the range of 0.3-0.8 A.U. If the absorbance of the wellsexceeded 1.5, they were washed and fresh Ellman' Reagent was added andthen redeveloped.

Calculation of Synergy and Combination Index

Synergy between the curcuminoids and andrographolide was assessed usingCalcuSyn (BIOSOFT, biosoft.com). This statistical package performsmultiple drug dose-effect calculations using the Median Effect methodsdescribed by T-C Chou and P. Talaly (Trends Pharmacol. Sci. 4:450-454),hereby incorporated by reference.

Briefly, it correlates the “Dose” and the “Effect” in the simplestpossible form: fa/fu=(C/Cm)m, where C is the concentration or dose ofthe compound and Cm is the median-effective dose signifying the potency.Cm is determined from the x-intercept of the median-effect plot. Thefraction affected by the concentration of the test material is fa andthe fraction unaffected by the concentration is fu (fu=1−fa). Theexponent m is the parameter signifying the sigmoidicity or shape of thedose-effect curve. It is estimated by the slope of the median-effectplot.

The median-effect plot is a plot of x=log (C) vs y=log (fa/fu) and isbased on the logarithmic form of Chou's median-effect equation. Thegoodness of fit for the data to the median-effect equation isrepresented by the linear correlation coefficient r of the median-effectplot. Usually, the experimental data from enzyme or receptor systemshave an r>0.96, from tissue culture an r>0.90 and from animal systems anr>0.85.

Synergy of test components is quantified using the combination index(CI) parameter. The CI of Chou-Talaly is based on the multipledrug-effect and is derived from enzyme kinetic models (Chou, T.-C. andTalalay, P. (1977) A simple generalized equation for the analysis ofmultiple inhibitions of Michaelis-Menten kinetic systems. J. Biol. Chem.252:6438-6442). The equation determines only the additive effect ratherthan synergism or antagonism. However, synergism is defined as a morethan expected additive effect, and antagonism as a less than expectedadditive effect as proposed by Cho and Talalay in 1983 (TrendsPharmacol. Sci. (1983) 4:450-454). Using the designation of CI=1 as theadditive effect, there is obtained for mutually exclusive compounds thathave the same mode of action or for mutually non-exclusive drugs thathave totally independent modes of action the following relationships:CI<1, =1, and >1 indicating synergism, additivity and antagonism,respectively.

Expected median inhibitory concentrations of the two-componentcombinations were estimated using the relationship:[1/Expected IC ₅₀ ]=[A/IC ₅₀ A]+[B/IC ₅₀ B]where A=mole fraction of component A in the combination and B=the molefraction of component B in the combination.

TABLE 3 illustrates the observed and expected median inhibitoryconcentrations for parthenolide and triptolide for PGE2 production byCOX-2 in the RAW 264.7 cell assay. While the expected IC₅₀ for the 1:2combination of parthenolide and triptolide was 0.131 μg/mL, the observedvalue was 0.032 μg/mL or 4.1-fold greater. This level of difference wasunexpected and constitutes a novel finding for the combined COX-2inhibitory activity of the 1:10 combination of parthenolide andtriptolide.

TABLE 3 Observed and Expected Median Inhibitory Concentrations for aFormulation of parthenolide and triptolide Combination ComponentsParthenolide Triptolide Expected Observed (1:2) IC₅₀ (μg/mL) IC₅₀(μg/mL) IC₅₀ (μg/mL) IC₅₀ (μg/mL) Parthenolide: 0.560 0.094 0.131 0.032Triptolide

Statistical analysis of inhibition of COX-2 production of PGE2 in theRAW 264.7 cell model for the 1:2 combination of parthenolide andtriptolide is presented in TABLE 4. The CI for this combination was0.250, 0.259 and 0.349, respectively, for the IC₅₀, IC₇₅ and IC₉₀. TheseCI values indicate strong synergy between parthenolide and triptolideover the complete dose-response curve.

TABLE 4 Combination Index for a 1:10 Formulation of parthenolide andtriptolide Combination Index IC50 IC75 IC90 Mean CI 0.250 0.295 0.3490.298

These data are consistent with and support the test results andconclusions performed in the Jurkat cells in which COX-2 proteinexpression was monitored.

Thus, there has been disclosed a formulation comprising triptolide andparthenolide. The combination formula provides for a synergisticanti-inflammatory effect in response to physical or chemical injury orabnormal immune stimulation due to a biological agent or unknownetiology.

It will be readily apparent to those skilled in the art that variouschanges and modifications of an obvious nature may be made withoutdeparting from the spirit of the invention, and all such changes andmodifications are considered to fall within the scope of the inventionas defined by the appended claims. Such changes and modifications wouldinclude, but not be limited to, the incipient ingredients added toaffect the capsule, tablet, lotion, food or bar manufacturing process aswell as vitamins, herbs, flavorings and carriers. Other such changes ormodifications would include the use of other herbs or botanical productscontaining the combinations of the present invention disclosed above.

1. A composition for inhibition of inducible cyclooxygenase-2 (COX-2)activity, said composition comprising as a first component an effectiveamount of a diterpene triepoxide lactone species, and as a secondcomponent an effective amount of a sesquiterpene lactone species,wherein the ratio of diterpene triepoxide lactone species tosesquiterpene lactone species is between 100:1 and 1:100.
 2. Thecomposition of claim 1 wherein the first and second components arederived from plants or plant extracts.
 3. The composition of claim 1wherein at least one of said first or second component is conjugatedwith a compound selected from the group consisting of mono- ordi-saccharides, amino acids, sulfates, succinate, acetate andglutathione.
 4. The composition of claim 1, formulated in apharmaceutically acceptable carrier.
 5. The composition of claim 1,additionally containing one or more members selected from the groupconsisting of antioxidants, vitamins, minerals, proteins, fats,carbohydrates, glucosamine, chondroitin sulfate and aminosugars.
 6. Acomposition for inhibition of inducible cyclooxygenase-2 (COX-2)activity, said composition comprising as a first component an effectiveamount of pharmaceutical grade compound selected from the groupconsisting of triptolide, triptonide, tripdiolide and tripchlorolide,and as a second component an effective amount of parthenolide, encelin,leucanthin B, enhydrin, melanomdin A, tenulin, confertiflorin, burrodin,psilostachyin A, costunolide, strigol, helenalin,5-α-hydroxy-dehydrocostuslactone, chlorochrymorin, chrysandiol,chrysartemin A, chrysartemin B, cinerenin, curcolone, cynaropicrin,dehydrocostus lactone, dehydroleucodin, dehydrozaluzanin C,deoxylatucin, eremanthine, eupaformonin, eupaformosanin, eupatolide,furanodienone, heterogorgiolide, lactucin, magnolialide, michelenolide,repin, spirafolide, and zaluzanin C.
 7. The composition of claim 6wherein the first and second components are derived from plants or plantextracts.
 8. The composition of claim 6 wherein at least one of saidfirst or second component is conjugated with a compound selected fromthe group consisting of mono- or di-saccharides, amino acids, sulfates,succinate, acetate and glutathione.
 9. The composition of claim 6,formulated in a pharmaceutically acceptable carrier.
 10. The compositionof claim 6, additionally containing one or more members selected fromthe group consisting of antioxidants, vitamins, minerals, proteins,fats, carbohydrates, glucosamine, chondrotin sulfate and aminosugars.11. A composition for inhibition of inducible cyclooxygenase-2 (COX-2)activity, said composition comprising as a first component an effectiveamount of pharmaceutical grade triptolide or triptonide, and as a secondcomponent an effective amount of a compound selected from the groupconsisting of parthenolide, encelin, leucanthin B, enhydrin, melapodinA, tenulin, confertiflorin, burrodin, psilostachyin A, costunolide,strigol and helenalin.
 12. The composition of claim 11 wherein the firstand second components are derived from plants or plant extracts.
 13. Thecomposition of claim 11 wherein at least one of said first or secondcomponent is conjugated with a compound selected from the groupconsisting of mono- or di-saccharides, amino acids, sulfates, succinate,acetate and glutathione.
 14. The composition of claim 11, formulated ina pharmaceutically acceptable carrier.
 15. The composition of claim 11,additionally containing one or more members selected from the groupconsisting of antioxidants, vitamins, minerals, proteins, fats,carbohydrates, glucosamine, chondrotin sulfate and aminosugars.
 16. Acomposition for inhibition of inducible cyclooxygenase-2 (COX-2)activity, said composition comprising as a first component an effectiveamount of pharmaceutical grade triptolide and as a second component aneffective amount of a compound selected from the group consisting ofparthenolide, encelin, leucanthin B, enhydrin, melapomdin A.
 17. Thecomposition of claim 16 wherein the first and second components arederived from plants or plant extracts.
 18. The composition of claim 16wherein at least one of said first or second component is conjugatedwith a compound selected from the group consisting of mono- ordi-saccharides, amino acids, sulfates, succinate, acetate andglutathione.
 19. The composition of claim 16, formulated in apharmaceutically acceptable carrier.
 20. The composition of claim 16,additionally containing one or more members selected from the groupconsisting of antioxidants, vitamins, minerals, proteins, fats,carbohydrates, glucosamine, chondrotin sulfate and aminosugars.
 21. Acomposition for inhibition of inducible cyclooxygenase-2 (COX-2)activity, said composition comprising as a first component an effectiveamount of pharmaceutical grade triptolide and as a second component aneffective amount of pharmaceutical grade parthenolide.
 22. Thecomposition of claim 21 wherein the first and second components arederived from plants or plant extracts.
 23. The composition of claim 21wherein at least one of said first or second component is conjugatedwith a compound selected from the group consisting of mono- ordi-saccharides, amino acids, sulfates, succinate, acetate andgluthathione.
 24. The composition of claim 21, formulated in apharmaceutically acceptahle carrier.
 25. The composition of claim 21,additionally containing one or more members selected from the groupconsisting of antioxidants, vitamins, minerals, proteins, fats,carbohydrates, glucosamine, chondrotin sulfate and aminosugars.